The alpha6beta4 integrin promotes epithelial cell proliferation and migration and may play a pivotal role in carcinoma invasion. These functions are mediated by the unique and large cytoplasmic domain of beta4. We have recently shown that alpha6beta4 palmitoylated and interacts with the doubly acylated Src kinases Fyn and Yes in lipid rafts. Ligand binding activates Fyn/Yes, which in turn phosphorylates the C-terminal half of the beta4 tail, causing recruitment of the signaling adaptor protein Shc and thereby activation of ERK and possibly PI-3K. Upon dephosphorylation, the beta4 tail mediates assembly of hemidesmosomes. Recent studies have indicated that alpha6beta4 also associates with the receptor protein tyrosine kinases (RPTKs) EGF-R, ErbB-21Neu, Met, and Ret, and their activated oncogenic versions. Strong evidence points to cooperative signaling by Alpha6beta4 and these RPTKs. Our major goal is to clarify the mechanisms by which alpha6beta4 promotes cell proliferation and migration/invasion and to identify the physiological and pathological processes regulated by alpha6beta4 signaling in vivo. In Aim 1, biochemical and microscopic methods will be used to examine the relationship between alpha6beta4 signaling and assembly of hemidesmosomes. In Aim 2, cell biological methods will be used to examine the mechanisms underlying cooperative signaling by alpha6beta4 and associated RPTKs and to identify the downstream pathways that promote cell proliferation, migration, and invasion. In Aim 3, the phenotype of mice carrying a targeted deletion of the signaling segment of the beta4 tail will be examined. Based on preliminary results, emphasis will be placed on the analysis of the signaling function of alpha6beta4 during postnatal angiogenesis. In Aim 4, mouse molecular genetics methods will be employed to study the role of alpha6beta4 in tumor invasion. The targeted deletion of the signaling domain of beta4 will be introduced in mice genetically engineered to develop carcinomas of the skin (K14-I-IPV16 and K14-SOS-F transgenic mice), mammary gland (MMTV-Neu and MT1-Trp-Met mice), and thyroid gland (TG-Ret/PTC3 mice). Analysis of tumor growth and invasion and tumor angiogenesis in these mice will reveal if alpha6beta4 signaling plays a role in these processes. By using both cell biology and genetics, we hope to achieve a better understanding of the signaling function of this important integrin.